Compounds and methods for the treatment of erb b2/neu positive diseases

ABSTRACT

Disclosed herein are anti-ERB B2/NEU antibodies conjugated with maytansinoid drugs for targeted delivery to disease tissues. Methods relating to the preparation and uses of such drug conjugates to treat ERB B2/NEU positive cells in cancers are provided.

FIELD OF INVENTION

The present invention generally relates to compounds comprisingantibodies, antigen-binding fragments thereof, polypeptides, andimmunoconjugates that bind to ERB B2/NEU (HER2). The present inventionalso relates to methods of using such ERB B2/NEU-binding molecules fordiagnosing and treating diseases, such as malignancies.

BACKGROUND OF INVENTION

The ErbB2/NEU (HER2) receptor is amplified and overexpressed in 20% to25% of human breast cancer and also predictive of poor clinical outcome,ErbB2/NEU (HER2) receptor was recognized and validated for targetedantibody therapy. Trastuzumab (Herceptin), a humanized anti-ErbB2/NEUreceptor antibody was approved by the Food and Drug Administration in1998 for use in metastatic breast cancer and later on for gastriccancer. Despite the clinical benefits, a significant proportion ofpatients treated with trastuzumab either do not respond initially orrelapse after experiencing a period of clinical response. There is needto develop more efficacious therapy. Direct covalent coupling ofcytotoxic agents to monoclonal antibodies is an alternative to nakedantibody-targeted therapy. Trastuzumab has been conjugated withmaytansine and tested in human clinical trials. However, it is knownthat different linkers, and the methods by which the linkers areconjugated with the antibody impact the efficacy and safety of the drugantibody conjugates.

Maytansinoids are highly cytotoxic compounds which inhibit the formationof microtubule protein polymerization (Remillard, et al., Science 189,1002-1005 (1975)). Maytansine was first isolated by Kupchan et al. (J.Am. Chem. Sci 94:1354-1356 (1972)) from the east African shrub Maytenusserrata. Maytansinoids including maytansinol and C-3 esters ofmaytansinol were also produced by certain microbes (U.S. Pat. No.4,151,042). Various analogues of maytansinol with different cytotoxicityhave also been prepared by synthetic chemistry (for review see Chem.Pharm. Bull. 52(1) 1-26 (2004)). Examples of mytansinoids includemaytansine, mertansine (MD1), MD3 and MD4. Maytansine is a strongmitotic inhibitor and shows significant inhibitory activity againstmultiple tumors including Lewis lung carcinoma and B-16 melanocarcinomasolid murine tumor models. Maytansine was reported to inhibit the humanacute lymphoblastic leukemia line C.E.M. at concentrations as low as10⁻⁷ □g/mL (Wolpert-DeFillippes et al., Biochem. Pharmacol. 1735-1738(1975)). It also showed to be 100- to 1000-fold more cytotoxic thanconventional chemotherapeutic agents like methotrexate, daunorubicin,and vincristine (U.S. Pat. No. 3,896,111).

Ansamitocins, the bacterial maytansinoids, show an activity spectrum andeffective dosage range similar to maytansine. They inhibit P388 leukemiaat daily doses as low as 0.8 □g/kg. Ansamitocin P3 (AP3) was also shownto be effective against multiple cancer cell lines (for review seeAlkaloids, vol. 2, 149-204 (1984); Chem. Pharm. Bull. 52(1) 1-26(2004)). The maytansinol C-3 esters with N-methyl-L-alanine derivativesare found to be much more cytotoxic than the corresponding esters ofsimple carboxylic acid and to be 100 times more cytotoxic than theirepimers corresponding to N-methyl-D-alanine (U.S. Pat. Nos. 4,137,230;4,260,608; Kawai, et al., Chem. Pharm. Bull. 32: 3441-3451 (1984);Widdison, et al., J. Med. Chem. 49: 4392-4408 (2006)).

Maytansinoids were expected to have the capacity to treat many differentcancers due to their highly toxic nature and the in vitro activitiesagainst multiple cancer cell lines. However, the toxicity also made thisclass of compounds not favorable in human clinical trials as the sideeffects were intolerable for many patients (Issel et al., 5 CancerTreat. Rev. 199-207 (1978)). Accordingly, targeted delivery of cytotoxiccompounds to cancer cells by conjugating toxic drugs to monoclonalantibodies (ADC for antibody drug conjugate) is proposed in order toreduce the side effects. Certain conjugates of cytotoxic drugs such asmaytansinoids, auristatins, anthracyclins, duocarmycins, etc. withantibodies are being evaluated in preclinical or clinical studies in thetreatment of diseases.

Antibody drug conjugates (ADCs) are composed of three key elements:antibody, linker, and drug. The selection of a particular antibody anddrug will have a great impact on the efficacy and safety depending onthe particular disease. Linker stability and the method by which thedrug is conjugated to the antibody plays a critical role in the successor failure of the ADC drug development.

The efficacy of an ADC depends in part on combination of a variety ofparameters, involving not only the specificity of the antibody and thepotency of drugs, but also the linker's stability or sensitivity tocleavage, the cell surface triggered the internalization, trafficking,and subsequent release of the active cytotoxic payload. Thus, ADCcomprising different drug linkers or with different antibodies againstthe same target can vary significantly in their utility.

SUMMARY OF THE INVENTION

The present invention provides an anti-Erb B2/neu antibody that isconjugated with maytansinoid molecules, thus targeting disease cells ortissues. The anti-Erb B2/neu antibody binds to an antigen in the diseasecells or tissues. A drug conjugated to the antibody exerts a cytotoxic,cytostatic, or immunosuppressive effect on the antigen-expressing cellsto treat or prevent recurrence of ERB B2/NEU-positive cancers. The highaffinity of the antibody drug conjugate ensure that the cytotoxicmaytansinoid targets the tumor cells. Otherwise, the highly toxicmaytansinoid will become systemically bound to unintended targets whichresults in very high and often unacceptable toxicity. The presenttechnology provides a method to treat cancers by exerting cellularinhibitory or killing effect of maytansinoid on the ERB B2/NEU positivecells, while minimizing the undesirable side effects of maytansinoid,such as bystander killing effects on antigen negative cells.

In one aspect, provided is an anti-Erb B2/neu antibody conjugated with amaytansinoid compound, wherein the maytansinoid compound is d linked toan anti-Erb B2/neu antibody via a linker that is not acid labile, notpeptidase cathepsin sensitive, and does not contain a disulfide bond andprovides stability during circulation while being able to release thedrug once inside the cells. Such linkers are contemplated to providestability to the conjugated molecule prior to endocytosis, such asduring circulation, to prevent premature degradation of the linker andrelease of the toxic drug, thus minimize the toxic effect of the drug.In some embodiments, the maytansinoid-linker portion of the conjugate isN2′-deacetyl-N2′-(6-maleimido-1-oxo-hexyl)-maytansine (3AA-MDC orbatansine), or a derivative thereof. In some embodiments, the conjugatehas a high drug load of at least 3 drug molecules per antibody forimproved activity. Surprisingly, the antibody remain sufficiently stablefor targeted delivery of the drug to target cells despite the high drugload.

In some embodiments, provided herein is a maytansinoid linker anti-ErbB2/neu antibody conjugate of Formula Ia or Ib:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;    -   p is selected from 3, 4, 5, 6, 7, 8, 9, 10; and    -   Anti-HER2 is anti-ERB B2/NEU antibody.

In another aspect, provided is a composition comprising theabove-described maytansinoid linker anti-Erb B2/neu antibody conjugate,such as a compound of Formula Ia.

In another aspect, provided is a method of preparing the above-describedmaytansinoid linker anti-Erb B2/neu antibody conjugate which methodcomprises contacting an anti-Erb B2/neu antibody with one or moremaytansinoid compounds described herein capable of being conjugated tothe anti-Erb B2/neu antibody.

In another aspect, provided is a method for targeting a maytansinoid toERB B2/NEU antigen positive cells or tissues with an anti-Erb B2/neuantibody conjugated with maytansinoids described herein.

In some embodiments, the anti-Erb B2/neu antibody has an amino acidsequence comprising the amino acid sequences of trastuzumab orpertuzumab, or an equivalent thereof.

In another aspect, provided is a method for treatment of proliferativedisorders such as tumors, inflammatory or immunologic diseases such asgraft rejections, and other diseases that can be treated by targetedtherapy in a subject in need of the treatment, wherein the disease ischaracterized by cells comprising an antigen that binds to an anti-ErbB2/neu antibody, said method comprising administering to the subject aneffective amount of the anti-Erb B2/neu antibody drug conjugatedescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of 3AA-MDC (the linkered drugN2′-deacetyl-N2′-(6-maleimido-1-oxo-hexyl)maytansine) and relatedmetabolites on the tubulin polymerization.

FIG. 2 shows anti-Erb B2/neu antibody and 3AA-MDC-antibody inhibitedtumor cell SK-BR-3 growth.

FIG. 3 shows Anti-Erb B2/NEU antibody or 3AA-MDC-Anti-ERB B2/NEUantibody has no inhibitory effect on Erb B2/NEU negative A549 negativecells.

FIG. 4 shows the inhibitory effect of D-Lmcc-Anti-Erb B2/neu antibodytowards SK-BR-3 cells.

FIG. 5 shows that Anti-Erb B2/NEU antibody or D-Lmcc-Anti-Erb B2/neuantibody has no inhibitory effect on ERB B2/NEU negative A549 cells.

FIG. 6 shows that 3AA-MDC-anti-HER2 (Erb B2/NEU) antibody eradicatedhuman BT474 tumor in mouse xenografts.

FIG. 7 shows that 3AA-MDC-anti-HER2 (Erb B2/NEU) antibody eradicatedhuman NCI-N87 tumor in mouse xenografts.

FIG. 8 shows a mass spectrum of 3AA-MDC, which was the metabolites of aprodrug the anti-Her2 antibody Cysteine-3AA-MDC

FIG. 9,10 shows a mass spectrum of two non enantiomers ofMDC-MCC-Lysine, which was the metabolites of D-Lmcc-anti-Her2 antibody.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “acompound” includes a plurality of compounds.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% or plus or minus 5%, orplus or minus 1% of the particular term.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

As used herein, “maytansinoid” refers to a maytansine analogue,including stereoisomers thereof. Maytansine can be isolated from plantsof the genus Maytenus U.S. Pat. No. 3,896,111). It is of the formula:

Maytansinoids are compounds having the ring structure of maytansine withone or more modifications of the substituents on the ring.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.C_(v) alkyl wherein v is an integer represents an alkyl having vcarbons. This term includes, by way of example, linear and branchedhydrocarbyl groups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl(CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—),n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—). “Alkylene” isa divalent saturated aliphatic hydrocarbyl groups having from 1 to 10carbon atoms and preferably 1 to 6 carbon atoms.

“Alkenyl” refers to straight or branched hydrocarbyl groups having from2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having atleast 1 and preferably from 1 to 2 sites of vinyl (>C═C<) unsaturation.Such groups are exemplified, for example, by vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of acetylenic (—C≡C—)unsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

“Amino” refers to the group —NR′R″ where R′ and R″ are independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, and wherein R′ and R″ areoptionally joined, together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group, provided that R′ and R″are both not hydrogen, and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. When R′ is hydrogen andR″ is alkyl, the substituted amino group is sometimes referred to hereinas alkylamino. When R′ and R″ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R′ and R″ is hydrogen butnot both. When referring to a disubstituted amino, it is meant thatneither R′ and R″ are hydrogen.

“Amino acid” refers any compound, whether natural, unnatural orsynthetic, which includes both an amino group and a carboxy group.Examples of amino acid include, but are not limited to glycine(NH₂CH₂COOH), cysteine, alanine, N-methyl-L-alanine, including both theD and L optical isomers. “Amino acid side chain” refers to thesubstituent that replaces a hydrogen of the methylene group of glycineor glycine derivatives, such as N-alkylglycine or glycine esters.Examples of an amino acid side chain include, but are not limited to theside chains of the natural amino acids, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heteroaryl, substituted heteroaryl,heterocyclic, and substituted heterocyclic.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Carbonyl” refers to the divalent group —C(O)— which is equivalent to—C(═O)—.

“Carboxy” or “carboxyl” refers to —COOH or CO₂H or salts thereof.

“Carboxylic acid” refers to a compound having at least one carboxy.

“Cyano” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. One or more of the rings can be aryl, heteroaryl, orheterocyclic provided that the point of attachment is through thenon-aromatic, non-heterocyclic ring carbocyclic ring. Examples ofsuitable cycloalkyl groups include, for instance, adamantyl,cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl. Other examples ofcycloalkyl groups include bicycle[2,2,2,]octanyl, norbornyl, andspirobicyclo groups such as spiro[4.5]dec-8-yl:

Cycloalkylene refers to a cyclic alkylene.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple cyclic rings and having atleast one >C═C<ring unsaturation and preferably from 1 to 2 sitesof >C═C<ring unsaturation.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is fluoro or chloro.

“Haloalkyl” refers to alkyl groups substituted with 1 to 5, 1 to 3, or 1to 2 halo groups, wherein alkyl and halo are as defined herein.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls includepyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or partially saturated, but not aromatic, grouphaving from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatomsselected from the group consisting of nitrogen, sulfur, or oxygen.Heterocycle encompasses single ring or multiple condensed rings,including fused bridged and spiro ring systems. In fused ring systems,one or more the rings can be cycloalkyl, aryl, or heteroaryl providedthat the point of attachment is through the non-aromatic ring. In oneembodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic groupare optionally oxidized to provide for the N-oxide, sulfinyl, orsulfonyl moieties.

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Substituted alkyl,” “substituted alkenyl,” “substituted alkynyl,”“substituted cycloalkyl,” “substituted cycloalkenyl,” “substitutedaryl,” “substituted heteroaryl” or “substituted heterocyclic” refers toalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl orheterocyclic groups, respectively, which are substituted with 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents selected fromthe group consisting of alkyl, halo alkyl, —O—R²⁰, —S—R²⁰, alkenyl,alkynyl, —C(═O)R²⁰, —C(═S)R²⁰, —C(═O)OR²⁰, —NR²⁰C(═O)R²¹, —OC(═O)R²¹,—NR²⁰R²⁰, —C(═O)NR²⁰R²⁰, —C(═S)NR²⁰R²⁰, —NR²⁰C(═O)NR²⁰R²⁰,—NR²⁰C(═S)NR²⁰R²⁰, —OC(═O)NR²⁰R²⁰, —SO₂NR²⁰R²⁰, —OSO₂NR²⁰R²⁰,—NR²⁰SO₂NR²⁰R²⁰, —C(═NR²⁰)NR²⁰NR²⁰R²⁰, aryl, —NR²⁰C(═O)OR²¹,—OC(═O)OR²¹, cyano, cycloalkyl, cycloalkenyl, —NR²⁰C(═NR²⁰)NR²⁰R²⁰,halo, hydroxy, heteroaryl, heterocyclic, nitro, —SO₃H, —SO₂R²¹, and—OSO₂R²¹, wherein each R²⁰ is selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl,heteroaryl, and heterocyclic or two R²⁰ with the atom(s) bound theretoform a heterocyclic ring, and R²¹ is selected from the group consistingof alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl,and heterocyclic.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O) or (—O⁻).

“Spiro ring systems” refers to bicyclic ring systems that have a singlering carbon atom common to both rings.

“Thiol” refers to the group —SH.

“Thiocarbonyl” refers to the divalent group —C(S)— which is equivalentto —C(═S)—.

“Thione” refers to the atom (═S).

“Compound” or “compounds” as used herein is meant to include thestereoisomers and tautomers of the indicated formulas.

“Stereoisomer” or “stereoisomers” refer to compounds that differ in thechirality of one or more stereocenters. Stereoisomers includeenantiomers and diastereomers.

“Tautomer” refer to alternate forms of a compound that differ in theposition of a proton, such as enol-keto and imine-enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a ring atomattached to both a ring —NH— moiety and a ring ═N— moiety such aspyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

“Solvate” refer to an association of a solvent with a compound, in thecrystalline form. The solvent association is typically due to use of thesolvent in the synthesis, crystallization, and/or recrystallization ofthe compound. “Solvate” includes hydrate which is an association ofwater with a compound, in the crystalline form.

“Patient” or “subject” refers to mammals and includes humans andnon-human mammals.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptablesalts of a compound, which salts are derived from a variety of organicand inorganic counter ions well known in the art and include, by way ofexample only, when the molecule contains an acidic functionality, saltsof organic or inorganic bases, such as sodium, potassium, calcium,magnesium, ammonium, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and tetraalkylammonium,and the like; and when the molecule contains a basic functionality,salts of organic or inorganic acids, such as hydrochloride,hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Othernon-limiting examples of acids include sulfuric acid, nitric acid,phosphoric acid, propionic acid, glycolic acid, pyruvic acid, malonicacid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicyclic acid and the like.

“Treating” or “treatment” of a disease in a patient refers to (1)preventing the disease from occurring in a patient that is predisposedor does not yet display symptoms of the disease; (2) inhibiting thedisease or arresting its development; or (3) ameliorating or causingregression of the disease.

“Effective amount” is intended to mean an amount of an active compoundor pharmaceutical agent that elicits the biological or medicinalresponse in a tissue, system, animal, individual or human that is beingsought by a researcher, veterinarian, medical doctor or other clinician,which includes treating a disease.

Anti-Erb B2/Neu Antibody Drug Conjugates

In one aspect, disclosed herein is a maytansinoid conjugated to ananti-ERB B2/NEU antibody via a linker that is not acid labile, notpeptidase cathepsin sensitive, and that is stable in circulation whilebeing able to release the cytotoxic drug inside the cells and with ahigh drug load of at least 3 drug molecules per antibody.

Maytansinoids suitable for attaching the linking group includemaytansinol and maytansinol analogues and can be isolated from naturalsources according to known methods, produced using biotechnologies (seee.g., Yu et al., 99 PNAS 7968-7973 (2002)), or prepared syntheticallyaccording to known methods (see e.g., Cassady et al., Chem. Pharm. Bull.52(1) 1-26 (2004)).

Certain examples of suitable maytansinol analogues include:

-   -   (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH        reduction of ansamytocin P2);    -   (2) C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat.        Nos. 4,361,650 and 4,307,016) (prepared by demethylation using        Streptomyces or Actinomyces or dechlorination using lithium        aluminium hydride (LAH));    -   (3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat.        No. 4,294,757) (prepared by acylation using acyl chlorides);    -   (4) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction        of maytansinol with H₂S or P₂S₅);    -   (5) C-14-hydroxymethyl (CH₂OH) or acyloxymethyl (CH₂OC(═O)phenyl        or CH₂C(═O)(C₁-C₅ alkyl)) (U.S. Pat. No. 4,331,598) (prepared        from Nocardia);    -   (6) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by        the conversion of maytansinol by Streptomyces);    -   (7) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929)        (isolated from Trewia nudlflora);    -   (8) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348)        (prepared by the demethylation of maytansinol by Streptomyces);        and    -   (9) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the        titanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinol can be useful as the linkage position,depending upon the type of linker. For example, for forming an esterlinkage, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with a hydroxylgroup and the C-20 position having a hydroxyl group are all suitable. Insome embodiments, the linkage position is the C-3 position.

In some embodiments, provided is a compound of Ia or Ib

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;    -   p is selected from 3, 4, 5, 6, 7, 8, 9, 10; and    -   Anti-HER2 is anti-ERB B2/NEU antibody.

In some embodiments, the compound of Ia is

or a pharmaceutically acceptable salt or solvate thereof, whereinAnti-Her2 is anti-ERB B2/NEU (HER2) antibody.

In some embodiments, the anti-Her2 antibody is trastuzumab or anequivalent thereof.

The maytansinoid component of the maytansinoid derivatives having alinking group capable of conjugating to an anti-Erb B2/neu antibody orthe maytansinoid linker anti-Erb B2/neu antibody conjugates can besubstituted by other suitable cytotoxic agents, for example, anauristatin, a DNA minor groove binding agent, a DNA minor groovealkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, apuromycin, a dolastatin, and a vinca alkaloid. Other suitable cytotoxicagents include anti-tubulin agents, such as an auristatin, a vincaalkaloid, a podophyllotoxin, a taxane, a baccatin derivative, acryptophysin, a maytansinoid, a combretastatin, or a dolastatin. In someembodiments, the cytotoxic agent is AFP, MMAF, MMAE, AEB, AEVB,auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16,camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B,nocodazole, colchicines, colcimid, estramustine, cemadotin,discodermolide, maytansine, DM-1, DM-3, DM-4, or eleutherobin. Suitableimmunosuppressive agents include, for example, gancyclovir, etanercept,cyclosporine, tacrolimus, rapamycin, cyclophosphamide, azathioprine,mycophenolate mofetil, methotrexate, cortisol, aldosterone,dexamethasone, a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor,or a leukotriene receptor antagonist. In some embodiments, the cytotoxicagent is AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel,docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,combretatstatin, chalicheamicin, maytansine, DM-1, DM-3, DM-4, ornetropsin.

The maytansinoid component of the maytansinoid derivatives having alinking group capable of conjugating to an anti-Erb B2/neu antibody andthe maytansinoid linker anti-Erb B2/neu antibody conjugates can also besubstituted by a suitable immunosuppressive agent, for example,gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin,cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate,cortisol, aldosterone, dexamethasone, a cyclooxygenase inhibitor, a5-lipoxygenase inhibitor, or a leukotriene receptor antagonist.

Anti-Erb B2/Neu Antibody

Anti-Erb B2/neu antibody include fragments of antibodies (polyclonal andmonoclonal) such as Fab, Fab′, F(ab′)₂, and Fv (see, e.g., Parham, J.Immunol. 131:2895-2902 (1983); Spring et al., J. Immunol. 113:470-478(1974); Nisonoff et al., Arch. Biochem. Biophys. 89:230-244 (1960));domain antibodies (dAbs) and antigen-binding fragments thereof,including camelid antibodies (see, e.g., Desmyter et al., Nature Struct.Biol, 3:752 (1996)); shark antibodies called new antigen receptors(IgNAR) (see, e.g., Greenberg et al., Nature, 374:168 (1995); Stanfieldet al. Science 305:1770-1773 (2004)).

Monoclonal antibody techniques allow for the production of anti-ErbB2/neu antibody in the form of specific monoclonal antibodies.Particularly well known in the art are techniques for creatingmonoclonal antibodies produced by immunizing mice, rabbits, or any othermammal with the antigen of interest such as the tumor specific antigensisolated from the target cell. Another method of creating anti-ErbB2/neu antibody is using phage libraries of scFv (single chain variableregion), specifically human scFv (see, e.g., Griffiths et al., U.S. Pat.Nos. 5,885,793 and 5,969,108; McCafferty et al., WO 92/01047; Liming etal., WO 99/06587), or domain antibodies using yeast selection system(see, e.g., U.S. Pat. No. 7,195,595). In addition, resurfaced antibodiessuch as those disclosed in U.S. Pat. No. 5,639,641 may also be used, asmay chimerized or humanized antibodies.

Selection of a particular anti-Erb B2/neu antibody is a matter of choicethat depends upon the disease type, cells and tissues that are to betargeted.

In some embodiments, the anti-Erb B2/neu antibody is human monoclonalantibody.

Anti-Erb B2/neu antibodies that have specificity to a tumor antigen canbe used. A “tumor antigen” as used herein, refers to an antigenicsubstance produced in tumor cells, i.e., it triggers an immune responsein the host. Tumor antigens are useful in identifying tumor cells andare potential candidates for use in cancer therapy. Normal proteins inthe body are not antigenic. Certain proteins, however, are produced oroverexpressed during tumorigenesis and thus appear “foreign” to thebody. This may include normal proteins that are well sequestered fromthe immune system, proteins that are normally produced in extremelysmall quantities, proteins that are normally produced only in certainstages of development, or proteins whose structure is modified due tomutation.

Anti-Erb B2/neu antibody having specificity to a protein that isoverexpressed on a tumor cell as compared to a corresponding non-tumorcell can also be used. ERB B2/NEU is highly expressed in a range ofsolid tumors including those of the breast and stomach.

It is contemplated that anti-Erb B2/neu antibody can be modified tointroduce an amino acid sequence having improved antibody-dependentcellular cytotoxicity (ADCC). For instance, amino acids in the Fc and/orhinge region can be modified to achieve improved ADCC. Examples ofIgG1-Fc that mediates improved ADCC, as well as methods of screening forsuch sequences, are known in the art (e.g., Stewart et al. Protein EngDes Sel. 24(9):671-8, 2011).

Conjugation of a Drug to an Anti-Erb B2/Neu Antibody

As discussed, a drug (e.g., a maytansinoid drug derivative) can beconjugated to an anti-Erb B2/neu antibody through a linker. In oneembodiment, the anti-Erb B2/neu antibody can be modified withappropriate bifunctional modifying agent. In some embodiments, a groupcomprising a thiol (SH) group (also referred to as thio-comprisinggroup) can be introduced to the side-chain of an amino acid residue,such as the side-chain of a lysine, on the anti-Erb B2/neu antibody. Forexample, the amino group of a lysine residue on the anti-Erb B2/neuantibody can be converted to a thiol-comprising group by reaction with2-iminothiolane (Traut's Reagent), or with N-succinimidyl3-(2-pyridyldithio)propanoate (SPDP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB), etc and followed by reduction witha reducing reagent, such as 2-mercaptoethanol, dithiothreitol (DTT) ortris(2-carboxyethyl)phosphine (TCEP).

Non-limiting examples of thiol-comprising group that can replace theside-chain amino group of a lysine residue include —NHC(═NH)(CH₂)_(n)SHand —NHC(O)(CH₂)_(n)SH, wherein n is 1, 2, 3, 4, 5 or 6. When athiol-comprising group is introduced to an amino acid residue, the aminoacid residue is referred to as thiolated amino acid. For example, whenthe side-chain amino group of a lysine residue is converted to athio-comprising group, the lysine residue is referred to as thiolatedlysine. The number of free thiol (SH) group introduced in an anti-ErbB2/neu antibody may vary, such as between 1 and about 20, or 5 to 15,and or 5 to 12. The linkers or drug-linkers can form bonds with the freethiol (SH) group of a thiolated lysine residue on the anti-Erb B2/neuantibody. In some embodiments, the number of linkers or drug-linkersthat form bonds with thiolated lysine residues in the anti-Erb B2/neuantibody is between 1 and about 10. In some embodiments, the number ofsuch formed bonds is at least 1, or alternatively at least 2, or 3, or4, or 5. In some embodiments, the number of such formed bonds is no morethan 10, or alternatively no more than 9, or 8, 7, 6, 5, or 4. In someembodiments, each anti-Erb B2/neu antibody, on average, is conjugatedwith 3-5 drug molecules.

In another embodiment, a drug-linker can be conjugated to an anti-ErbB2/neu antibody by binding to the thiol group of a cysteine residue.Each anti-Erb B2/neu antibody typically contains multiple cysteines, butmany, if not all, of them form disulfite bonds between each other, andthus are not available for such conjugation. In some embodiments,therefore, one or more of the disulfite bonds of the anti-Erb B2/neuantibody can be broken to form free thiol (SH) groups by reaction with areducing reagent, such as 2-mercaptoethanol, dithiothreitol (DTT) ortris(2-carboxyethyl)phosphine (TCEP), for instance. The reaction can bemonitored and/or controlled so that a sufficient number of disulfitebonds are broken to allow conjugation while maintaining a sufficientnumber of disulfide bonds to keep the structure stability of theanti-Erb B2/neu antibody.

In some embodiments, the number of bonds formed between the drug-linkerand cysteine residue on the anti-Erb B2/neu antibody is from 3 to 10. Inone embodiment, the number of such bonds is at least 3, or alternativelyat least 4, or 5. In some embodiments, the number of such formed bondsis no more than 10, or alternatively no more than 9, or 8, 7, 6, 5, or4. In one embodiment, each anti-Erb B2/neu antibody, on average, isconjugated with 3-5 drug molecules through cysteines.

In some embodiments, drug molecules are conjugated to the anti-ErbB2/neu antibody through a mixture of lysine and cysteine residues.

An anti-Erb B2/neu antibody can be modified, by way of, e.g.,site-specific mutagenesis, to introduce additional thiolated lysine orcysteine residues to allow suitable conjugation. Amino acid modificationmethods are well known in the art. Modified anti-Erb B2/neu antibody canthen be experimentally examined for their stability and antigen bindingcapability. In one embodiment, at least one thiolated lysine or cysteineresidue is introduced by such modification. In another embodiment, atleast two thiolated lysine or cysteine residues are introduced by suchmodification.

Drug Load

The drug load on an anti-Erb B2/neu antibody may vary depending on manyfactors, such as the potency of the drug, the size, stability of theanti-Erb B2/neu antibody, conjugatable groups available on the anti-ErbB2/neu antibody, etc. In some embodiments, 1 to 10 maytansinoid drugmolecules are conjugated with 1 anti-Erb B2/neu antibody molecule. Insome embodiments, an average of 3 to 5 maytansinoid drug molecules areconjugated with 1 anti-Erb B2/neu antibody molecule. In someembodiments, an average of 3.5 maytansinoid drug molecules areconjugated with 1 anti-Erb B2/neu antibody molecule.

Metabolites of Maytansinoids-Linker-Anti-Erb B2/Neu Antibody Conjugatesto Release the Effective Agents

While not wishing to be bound to any theories, it is contemplated thatupon endocytosis, compounds of any one of Formula Ia-Ic is degraded byintracellular proteins to metabolites comprising the maytansinoid moietywhich are cytotoxic. In some embodiments, the compound is of FormulaIVa, IVb or IVc:

or a salt thereof, wherein AA is selected from, but is not limited to

wherein

represents point of connection to the rest of the molecule, and othervariables are as defined herein.

Methods of Treatment

In another aspect, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofone or more compounds as described herein, for example, a compound ofany one of Formula Ia-Ic and IVa-IVc-.

The compounds can be formulated as pharmaceutical compositions andadministered to the patient in a variety of forms adapted to the chosenroute of administration, i.e., orally or parenterally, by intravenous(I.V.), intramuscular, topical or subcutaneous routes. The amount of thecompounds will vary depend on the nature of the drug, linker, drug load,degree of cell surface triggered the internalization, trafficking, andrelease of the drug, the disease being treated, the conditions of thepatient, such as age, gender, weight, etc. and can be determined bymethods known to the art, for example, see U.S. Pat. No. 4,938,949, andwill be ultimately at the discretion of the attendant physician orclinician.

In general, a suitable dose will be in the range of from about 0.1 toabout 200 mg/kg, e.g., from about 0.5 to about 50 mg/kg of body weightI.V. infusion over 30-90 min every 1-4 week for 52 weeks, about 1.0 toabout 25 mg/kg of body weight IV infusion over 30-90 min every 1-4 weekfor 52 weeks, about 1.5 to about 15 mg/kg body weight IV infusion over30-90 min every 1-4 week for 52 weeks, or in the range of about 1 to 10mg/kg body weight IV infusion over 30-90 min every 1-4 week. In someembodiments, the dose is from about 1.0 mg to about 100 mg/day, e.g.,from about 2 mg to about 5 g per day, about 10 mg to about 1 g per day,about 20 to about 500 mg per day, or in the range of about 50 to 100 mgper day. The compounds can be administered daily, weekly, monthly, suchas once a day, every 1-3 weeks, or month. Alternatively, the compoundscan be administered in cycles, such as administered daily for a numberof days, for example, 5 days to 21 days, with a period, such as one dayto seven days, wherein no drug is being administered.

In some embodiments, the compound is administered at an initial dose of1-4 mg/kg over 30-90 minute IV infusion, followed by 1-2 mg/kg over 30minute I.V. infusion weekly or every 1-4 weeks for 52 weeks. In someembodiments, the compound is administered at an initial dose of 2-10mg/kg over 30-90 minutes I.V. infusion, followed by 1-5 mg/kg over 30-90minutes IV infusion every 1-4 weeks for 52 weeks.

In some embodiments, the compounds are administered in conjunction withanother therapy. For example, the compounds can be co-administered withanother therapy for treating cancer, for example, radiation therapy oranother anticancer agent known in the art.

In another aspect, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula IVa, wherein the compound of Formula IVa isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula Ia, or a pharmaceuticallyacceptable salt thereof, to the patient.

In another aspect, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula IVb, wherein the compound of Formula IVb isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula Ib, or a pharmaceuticallyacceptable salt thereof, to the patient.

In another aspect, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula IVc, wherein the compound of Formula IVc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula Ic, or a pharmaceuticallyacceptable salt thereof, to the patient.

Metabolic chemical reaction refers to a reaction occurring inside thebody, for example, cells, of the subject, in which a chemical compoundis converted to another chemical compound. The conversion can be bymetabolic and/or chemical processes and can occur in one step or througha series of two or more steps. Metabolic chemical reactions includereactions of degrading a protein or peptide component of a maytansinoidlinker anti-Erb B2/neu antibody conjugate, such as an antibody orantibody fragment, by proteins inside a cell.

Pharmaceutical Compositions

In a further aspect, provided are pharmaceutical compositions comprisingone or more compounds as described herein, for example, a compound ofany one of Formula Ia-Ic, and one or more pharmaceutically acceptablecarriers. Such compositions should contain at least 0.1% of activecompound. The percentage of the compositions may vary and may be betweenabout 2 to about 90% of the weight of a given unit dosage form. Theamount of active compound in such therapeutically useful compositions issuch that an effective dosage level will be obtained.

Examples of pharmaceutical compositions suitable for injection orinfusion can include sterile aqueous solutions or dispersions in apharmaceutically acceptable liquid carrier or vehicle, or sterilepowders comprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. Other forms ofpharmaceutical compositions include topical formulations, such as gel,ointments, creams, lotions or transdermal patches, etc. Thepharmaceutical compositions include using techniques well known to thosein the art. Suitable pharmaceutically-acceptable carriers, outside thosementioned herein, are known in the art; for example, see Remington, TheScience and Practice of Pharmacy, 20th Edition, 2000, LippincottWilliams & Wilkins, (Editors: Gennaro, A. R., et al.).

In a further aspect, provided are methods of producing a pharmaceuticalcomposition comprising admixing a compound as described herein, forexample, a compound of any one of Formula Ia-IVc, and a pharmaceuticallyacceptable carrier. Methods of admixing an active ingredient with apharmaceutically acceptable carrier are generally known in the art, forexample, uniformly mixing the active compound(s) with liquids or finelydivided solid carriers, or both, in the required proportions, and then,if necessary, forming the resulting mixture into a desired shape.

In some embodiments, a compound of any one of Formula Ia-IVc isformulated as an injectable, for example, at a concentration of 2-50mg/mL in an aqueous solution comprising 4-10 mg/mL sodium chlorideand/or 5-12 mg/mL sodium acetate, or alternatively at a concentration of2-50 mg/mL in an aqueous solution comprising 5-10 mg/mL sodium chloride,1-5 mg/mL sodium phosphate dibasic heptahydrate, 0.1-0.5 mg/mL sodiumphosphate monobasic monohydrate.

Other examples of formulations of a compound of any one of FormulaIa-IVc include an injectable formulation having a concentration of 2-100mg/mL of the compound in an aqueous solution comprising 0.5-1.0% sodiumchloride, 0.05-0.10% monobasic sodium phosphate dihydrate, 1.0-2.0%dibasic sodium phosphate dihydrate, 0.01-0.05% sodium citrate,0.10-0.20% citric acid monohydrate, 1.0-2.0% mannitol, 0.1%-0.2polysorbate 80, and Water for Injection, USP. Sodium hydroxide added asnecessary to adjust pH.

Methods

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc) are given, other process conditionscan also be used unless otherwise stated. Optimum reaction conditionsmay vary with the particular reactants or solvent used, but suchconditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

Furthermore, the compounds of this invention may contain one or morechiral centers. Accordingly, if desired, such compounds can be preparedor isolated as pure stereoisomers, i.e., as individual enantiomers ordiastereomers, or as stereoisomer-enriched mixtures. All suchstereoisomers (and enriched mixtures) are included within the scope ofthis invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

The starting materials for the following reactions are generally knowncompounds or can be prepared by known procedures or obviousmodifications thereof. For example, many of the starting materials areavailable from commercial suppliers such as Aldrich Chemical Co.(Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce orSigma (St. Louis, Mo., USA). Others may be prepared by procedures, orobvious modifications thereof, described in standard reference textssuch as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15(John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds,Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989),Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March'sAdvanced Organic Chemistry, (John Wiley and Sons, 4^(th) Edition), andLarock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989).

The various starting materials, intermediates, and compounds of theinvention may be isolated and purified where appropriate usingconventional techniques such as precipitation, filtration,crystallization, evaporation, distillation, and chromatography.Characterization of these compounds may be performed using conventionalmethods such as by melting point, mass spectrum, nuclear magneticresonance, and various other spectroscopic analyses.

Coupling reagents include carbodiimide, amininum and phosphonium basedreagents. Carbodiimide type reagents include dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC), and1-ethyl-3-(3-dimethylaminopropyl)-dicarbodiimide (EDC), etc. Aminiumsalts includeN-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HATU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HBTU),N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HCTU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TBTU), andN-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TCTU). Phosphonium salts include7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate (PyAOP) andbenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP). Amide formation step may be conducted in a polar solvent suchas dimethylformamide (DMF) and may also include an organic base such asdiisopropylethylamine (DIEA) or dimethylaminopyridine (DMAP).

For example, compounds of Formula Ia or Ib can be prepared by contactinga compound of Formula A or B, respectively, wherein the variables are asdefined herein, with an antibody in a suitable solvent, such as abuffer.

The following examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in practicing theinvention. These examples are in no way to be considered to limit thescope of the invention.

Example 1 Esterification of Maytansinol with Fmoc-N-methyl-L-alanine(Fmoc-N-Me-D/L-Ala-MDC)

A mixture of maytansinol (0.600 g, 1.062 mmol), Fmoc-N-Me-L-Ala (6.911g, 21.24 mmol), Sc(OTf)₃ (0.314 g, 0.637 mmol) and DMAP (0.389 g, 3.186mmol) in CH₂Cl₂ (100 mL) was stirred for 0.5 h at −8° C. DIC (2.949 g,23.37 mmol) was added dropwise, stirred for 0.5 h, warmed to r.t.slowly, filtered to recover the Lewis acid catalyst, the filtrate wasquenched with diluted HCl and extracted with CH₂Cl₂. The combinedorganic phase was washed with NaHCO₃ aq, brine, dried over anhydrousNa₂SO₄. The solvent was removed under reduced pressure. Chromatography(silica gel, CH₂Cl₂/MeOH 30:1) gave the desired product as a mixture ofdiastereomer Fmoc-N-Me-D/L-Ala-MDC: white solid (0.8385 g, 90.5%).Further column chromatography (silica gel, CH₂Cl₂/MeOH 100:1 to 20:1)gave two fractions as pure diastereomer. The higher Rf fraction wasdetermined to be the D-aminoacyl ester diastereomer(Fmoc-N-Me-D-Ala-MDC), while the lower Rf fraction was the desiredL-aminoacyl ester (Fmoc-N-Me-L-Ala-MDC). Fmoc-N-Me-L-Ala-MDC: whitesolid (0.4262 g, 46.0% yield), ¹H NMR (400 MHz, CDCl₃): δ0.77 (3H, s),1.22-1.32 (6H, m), 1.40-1.48 (1H, m), 1.63 (3H, s), 2.13 (1H, dd,J=14.4, 2.8 Hz), 2.53 (1H, dd, J=14.4, 10.8 Hz), 2.64 (3H, s), 2.88 (3H,s), 3.00 (1H, d, J=9.6 Hz), 3.07 (1H, d, J=12.4 Hz), 3.35 (3H, s), 3.48(1H, d, J=8.8 Hz), 3.59 (1H, d, J=11.2 Hz), 3.97 (3H, s), 4.13-4.19 (1H,m), 4.15 (1H, s), 4.24 (1H, t, J=10.8 Hz), 4.72-4.77 (2H, m), 5.03 (1H,q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.29 (1H, br), 6.41 (1H,dd, J=15.2, 11.2 Hz), 6.52 (1H, d, J=1.2 Hz), 6.70 (1H, d, J=10.8 Hz),6.79 (1H, d, J=1.2 Hz), 7.33 (1H, t, J=7.6 Hz), 7.36 (1H, t, J=7.6 Hz),7.39 (1H, d, J=7.6 Hz), 7.49 (1H, d, J=7.6 Hz), 7.70 (1H, d, J=7.6 Hz),7.72 (1H, d, J=7.6 Hz). LC-MS (M+Na⁺) calc.: 894.3, found: 894.3.Fmoc-N-Me-D-Ala-MDC: white solid (0.3993 g, 43.1% yield), ¹H NMR (400MHz, CDCl₃): δ0.84 (3H, s), 1.22-1.27 (3H, m), 1.40-1.48 (1H, m), 1.51(3H, d, J=7.6 Hz), 1.67 (3H, s), 2.20 (1H, dd, J=14.4, 2.8 Hz), 2.63(1H, dd, J=14.4, 12.4 Hz), 2.85 (1H, d, J=9.6 Hz), 2.96 (3H, s), 3.17(3H, s), 3.20 (1H, s), 3.24 (3H, s), 3.40 (1H, d, J=9.2 Hz), 3.51 (1H,d, J=12.8 Hz), 3.99 (3H, s), 4.20-4.28 (2H, m), 4.38-4.43 (2H, m),4.80-4.98 (2H, m), 5.80 (1H, dd, J=15.2, 11.2 Hz), 6.18 (1H, s), 6.25(1H, d, J=10.8 Hz), 6.40 (1H, dd, J=15.2, 11.2 Hz), 6.79 (1H, d, J=1.6Hz), 6.84 (1H, d, J=1.6 Hz), 7.32 (2H, t, J=7.6 Hz), 7.41 (2H, t, J=7.6Hz), 7.61 (2H, d, J=7.6 Hz), 7.77 (2H, d, J=7.6 Hz). LC-MS (M+Na⁺)calc.: 894.3, found: 894.3.

Example 2 Deprotection of Fmoc-N-Me-D/L-Ala-MDC (N-Me-D/L-Ala-MDC)

Into Fmoc-N-Me-D/L-Ala-MDC (0.463 g, 0.5307 mmol) in ACN (200 mL) wasadded piperidine (0.865 g, 10.15 mmol). The mixture was stirred at r.t.for 4 h, quenched with water and extracted with CH₂Cl₂. The combinedorganic phase was washed with brine and dried over Na₂SO₄. The solventwas removed under reduced pressure to give the crude product, which wasused in the next step without further purification. LC-MS (M+H⁺) calc.:650.3, found: 650.3. Rt: 3.96 min.

Example 3 Deprotection of Fmoc-N-Me-L-Ala-MDC (N-Me-L-Ala-MDC)

Into Fmoc-N-Me-L-Ala-MDC (0.463 g, 0.5307 mmol) in ACN (200 mL) wasadded piperidine (0.865 g, 10.15 mmol). The mixture was stirred at r.t.for 4 h, quenched with water and extracted with CH₂Cl₂. The combinedorganic phase was washed with brine and dried over Na₂SO₄. The solventwas removed under reduced pressure to give the crude product, which wasused in the next step without further purification. LC-MS (M+H⁺) calc.:650.3, found: 650.3. Rt: 3.96 min.

Example 4 Condensation of N-Me-D/L-Ala-MDC with MA-ACP (D-3AA-MDC andL-3AA-MDC)

Into above prepared N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g,2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123mmol). The mixture was stirred at r.t. overnight, quenched with water,extracted with EtOAc, washed with brine, and dried over Na₂SO₄. Thesolvent was removed under reduced pressure. Chromatography (silica gel:CH₂Cl₂/MeOH 30:1) gave the crude product. Further purification bypreparative HPLC on a YMC C-18 column (250×20 mm, S 10 μm) gave twofractions (Rt=6.59 min and 6.98 min) as white solid. The higher Rtfraction was determined to be the D-aminoacyl ester diastereomer(D-3AA-MDC, 45.2%), while the lower Rt fraction was the desiredL-aminoacyl ester (L-3AA-MDC, 54.8%). L-3AA-MDC: white solid (0.1364 g,30.5% overall yield over two steps), ¹H NMR (400 MHz, CDCl₃): δ0.79 (3H,s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m),2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H,s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34(3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d,J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H,s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72(1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na⁺) calc.: 865.3, found:865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2% overallyield over two steps), ¹H NMR (400 MHz, CDCl₃): δ0.86 (3H, s), 1.22-1.38(4H, m), 1.25 (3H, d, J=9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J=7.6Hz), 1.56-1.70 (4H, m), 1.68 (3H, s), 1.75 (1H, d, J=13.6 Hz), 2.19 (1H,dd, J=14.4, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J=14.2, 12.0 Hz),2.80 (1H, d, J=9.6 Hz), 3.01 (3H, s), 3.19 (1H, d, J=13.2 Hz), 3.32 (3H,s), 3.42 (1H, d, J=9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H,t, J=10.4 Hz), 4.88 (1H, dd, J=11.8, 3.2 Hz), 5.07 (1H, q, J=7.6 Hz),5.84 (1H, dd, J=15.2, 9.2 Hz), 6.23 (1H, d, J=11.2 Hz), 6.27 (1H, s),6.41 (1H, dd, J=15.2, 11.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J=1.2 Hz),6.84 (1H, d, J=1.2 Hz). LC-MS (M+Na⁺) calc.: 865.3, found: 865.3. Rt:6.98 min.

Example 5 Condensation of N-Me-L-Ala-MDC with MA-ACP (L-3AA-MDC)

Into above prepared N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g,2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123mmol). The mixture was stirred at r.t. overnight, quenched with water,extracted with EtOAc, washed with brine, dried over Na₂SO₄. The solventwas removed under reduced pressure. Chromatography (silica gel:CH₂Cl₂/MeOH 30:1) gave the crude product. Further purification bypreparative HPLC on a YMC C-18 column (250×20 mm, S 10 μm) gave thedesired L-3AA-MDC: white solid (0.280 g, 62.6% overall yield over twosteps), ¹H NMR (400 MHz, CDCl₃): δ0.79 (3H, s), 1.17-1.32 (3H, m), 1.27(3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45(1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz),3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27(1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz),5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s).LC-MS (M+Na⁺) calc.: 865.3, found: 865.3. Rt: 6.59 min.

Example 6 The Effect of the Metabolites of Prodrug Antibody MaytansinoidConjugates on the Tubulin Polymerization

The effect of 3AA-MDC, 206-3AA-MDC and the metabolites (Cys-3AA-MDC andLys-mcc-MDC) of prodrug antibody maytansinoid conjugates on the tubulinpolymerization in vitro was assessed by HTS-Tubulin Polymerization AssayKit (BK004P, Cytoskeleton, Inc., USA). According to the instruction ofkit, pre-warm the 96-well plate to 37° C. for 30 min prior to startingthe assay. At the same time, the spectrophotometer (SpectraMax,Molecular Devices, USA) was set as follow: wavelength, 405 nm;temperature, 37° C.; Kinetic, 31 cycles of 1 reading per minute. Makecold G-PEM buffer (990 μL General Tubulin Buffer+10 μL GTP Stock) andkeep it on ice. Prepare 4 mg/mL tubulin, 1 μM L-3AA-MDC(N₂′-deacetyl-N₂′-(6-maleimido-1-oxo-hexyl)maytansine), 1 μM206-3AA-MDC, 1 μM cys-3AA-MDC, 1 μM lys-mcc-MDC, 100 μM Paclitaxel, and100 μM Nocodazole. Add 10 μL G-PEM, 3AA-MDC, 206-3AA-MDC, cys-3AA-MDC,lys-mcc-MDC, Paclitaxel, Nocodazole into the wells, and then add 100 μL4 mg/ml tubulin to each well. Immediately place the plate into thespectrophotometer and start recording using the kinetic setup describedabove. As show in the FIG. 6, compared with the PBS buffer, 3AA-MDC,Cys-3AA-MDC, Lys-mcc-MDC and 206-3AA-MDC more significantly inhibitedthe tubulin polymerization (FIG. 6). Nocodazole, the tubulinpolymerization inhibitor, was set as a negative control. The metaboliteCys-3AA-MDC was prepared by reaction of 3AA-MDC with cysteine under thebase DIEA in CH₂Cl₂. LC-MS (M+H⁺) calc.: 964.5, found: 964.2. Rt: 12.97min. The metabolite Lys-MCC-MDC was prepared by reaction of SMCC-MDCwith lysine under the base DIEA in DMF. LC-MS (M+H⁺) calc.: 1103.7,found: 1103.2. Rt: 13.00 and 13.18 min.

Example 7 The Effect of 3AA-MDC and Related Metabolites on the TubulinPolymerization

The effect of 3AA-MDC and related metabolites on the tubulinpolymerization in vitro was assessed by HTS-Tubulin Polymerization AssayKit (BK004P, Cytoskeleton, Inc., USA). According to the instruction ofkit, pre-warm the 96-well plate to 37° C. for 30 min prior to startingthe assay. At the same time, the spectrophotometer (SpectraMax,Molecular Devices, USA) was set as follow: wavelength, 405 nm;temperature, 37° C.; Kinetic, 31 cycles of 1 reading per minute. Makecold G-PEM buffer (990 μL General Tubulin Buffer+10 μL GTP Stock) andkeep it on ice. Prepare 4 mg/ml tubulin, 200 nM L-3AA-MDC(N2′-deacetyl-N2′-(6-maleimido-1-oxo-hexyl)maytansine), or relatedcompounds cys-3AA-MDC, or lys-mcc-MDC, 100 μM Paclitaxel, 100 μMNocodazole, and 1 μM 206-3AA-MDC (3AA-MDC-antibody). Add 10 μL G-PEM,3AA-MDC, Paclitaxel, Nocodazole and 206-3AA-MDC or related metabolitesinto the wells, and then add 100 μL 4 mg/ml tubulin to each well.Immediately place the plate into the spectrophotometer and startrecording using the kinetic setup described above. As show in the FIG.1, the relative activity of tubulin polymerization was enhanced about 3fold in the presence of Paclitaxel and decreased 2.5 fold in thepresence of Nocodazole. L-3AA-MDC and other related metabolites moresignificantly inhibited the tubulin polymerization (FIG. 1).

Example 8 Recombinant Antibody Expression and Purification

The anti-ERB B2/NEU antibody was produced in CHO cells essentially asdescribed in Wood et al., J Immunol. 145:3011 (1990). Briefly, each ofthe antibody genes were constructed with molecular biology techniques(Molecular Cloning: A Laboratory Manual, 3^(rd) edition J. Sambrook etal., Cold spring Harbor Laboratory Press). A derivative of Chinesehamster ovary cell lines CHOK1 was grown in CD-CHO media (GIBCO).Transfections were facilitated using electroporation. Healthy mid-logCHO-K1 cells were pelleted by centrifuge and were resuspended in freshCD-CHO media to achieve cell densities of approximately 1×10⁷ cells (600mL) per cuvette. Suspensions of cells containing 40 μg of linearizedplasmid DNA were electroporated, seeding 10³ cells per well in 96-welltissue culture plates containing suitable selection drug. The antibodyexpression level in the culture supernatant of clones isolated on96-well tissue culture plates was determined by an enzyme-linkedimmunosorbent assay (ELISA). On the basis of the antibody titer in thesupernatant, clones with high-level expression were transferred to24-well plate (Corning) containing suitable media. Specific antibodyproductivity (qAb) and specific growth rate (μ) were further analyzed byseeding cells at 2×10⁵ cells per well containing 5 mL of medium insix-well tissue culture plates, culturing for 2 and 4 days, and usually20-30 high-producing clones (parental clones) were transferred to shakeflask for successive selection, and 5-8 highest producer clones werechosen to be further subcloned, and tested for expression.

The purification was carried out by centrifuging cell suspension andharvesting the supernatant, which was further cleared by centrifuging.Protein A affinity columns such as Mab Select SuRe (GE Healthcare) andion exchange such as Capto S (GE) were used to purify the expressedantibodies).

Example 9 Conjugation of Anti-Erb B2/Neu Antibody with SMCC-MDC

The drug-linker SMCC-MDC was prepared in the following reactions: (1)3-mercaptopropanoic acid (MPr) was reacted with N-succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) in the presence ofN,N-diisopropylethylamine (DIEA), giving the MPr-SMCC at a yield of over95%; (2) condensation of N-Me-L-Ala-MDC, which was prepared bydeprotection of Fmoc-N-Me-Ala-MDC under a base piperidine in CH₃CN, withMPr-SMCC under a coupling reagent EDC, giving the desired coupledproduct SMCC-MDC in 60-70% yield over two steps. Anti-Erb B2/neuantibody was diluted to 2.5 mg/mL in solution A (50 mM potassiumphosphate, 50 mM NaCl, and 2 mM EDTA, pH 6.5). SMCC-MDC was added togive a ratio of SMCC-MDC to antibody of 7:1 mole equivalent. Then DMA(dimethylacetamide) was added to 15% (v/v) to the reaction and reactionwas mixed by stirring for 4 h at ambient temperature. D-Lmcc-Anti-ErbB2/neu antibody conjugate was purified from excess unreacted orhydrolyzed reagent and excess SMCC-MDC using a G25 gel filtration columnequilibrated in pH 7.4 phosphate buffer (aqueous). The conjugate wasthen dialyzed overnight into pH 7.4 phosphate buffer (aqueous) and thenfiltered through a 0.22 □m filter for final storage. The number ofSMCC-MDC molecule per antibody molecule in the final conjugate wasmeasured by determining absorbance of the conjugate at 252 and 280 nmand using known extinction coefficients for SMCC-MDC and antibody atthese two wavelengths. A ratio of maytansinoid compound to antibody of3.5:1.0 was normally obtained.

Example 10 Conjugation of Anti-Erb B2/Neu Antibody with DerivatizedMaytansinoid

Anti-Erb B2/neu antibody was diluted to 8.0 mg/mL in solution B (50 mMpotassium phosphate, 50 mM NaCl, and 2 mM EDTA, pH 8.0). Partialreduction was carried out with (6 moles equivalent) DTT. Afterincubation at 37° C. for 60 minutes, the buffer was exchanged by elutionthrough Sephadex G-25 resin with solution B. The thiol-antibody valuewas determined from the reduced monoclonal antibody (mAb) concentrationdetermined from 280-nm absorbance, and the thiol concentration wasdetermined by reaction with DTNB (5,5′-dithiobis(2-nitrobenzoic acid);Aldrich) and absorbance measured at 412 nm.

The conjugation reaction was carried out with 10% DMA(dimethylacetamide). The volume of 3AA-MDC (the linked drugN2′-deacetyl-N2′-(6-maleimido-1-oxo-hexyl)maytansine) solution wascalculated to contain 1.5-mol 3AA-MDC per mol thiol equivalent. 3AA-MDCsolution was added rapidly with mixing to the cold-reduced antibodysolution, and the mixture was stirred at r.t. for 3 hours, and continuedfor additional 1 h after adding 5 mM cysteine. The reaction mixture wasconcentrated by centrifugal ultrafiltration and buffer-exchanged byelution through Sephadex G25 equilibrated in PBS. The conjugate was thenfiltered through a 0.2-1 μm filter under sterile conditions and storedat −80° C. for analysis and testing. The 3AA-MDC-antibody was furtheranalyzed for drug/antibody ratio by measuring unreacted thiols withDTNB, and 3.5:1 ratio of drug/antibody was often obtained.3AA-MDC-antibody was further characterized for concentration by UVabsorbance, aggregation by size-exclusion chromatography, and residualfree drug by reverse-phase HPLC. All mAbs and ADCs used in these studiesexceeded 98% monomeric protein.

Example 11 Preparation of Conjugated D-LSPP-Anti-Erb B2/Neu Antibody

Anti-Erb B2/neu antibody (8 mg/mL) was modified using 8-fold molarexcess of N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) tointroduce dithiopyridyl groups. The reaction was carried out in 95% v/vBuffer A (50 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5) and5% v/v dimethylacetamide (DMA) for 2 h at room temperature. The slightlyturgid reaction mixture was gel-filtered through a Sephadex G25 column(equilibrated in Buffer A). The degree of modification was determined bymeasuring the absorbance of the antibody and the 2-mercaptopyridine(Spy) released by DTT respectively at 280 and 343 nm. Modified anti-ErbB2/neu antibody was then conjugated at 2.5 mg/mL using a 1.7-fold molarexcess of NT-deacetyl-N-2′(3-mercapto-1-oxopropyl)-maytansine over SPy.The reaction was carried out with DMA (5% v/v) in Buffer A (see above).The reaction was incubated at room temperature overnight for 17 h. Theconjugated antibody was cleared by centrifugation and then furtherpurified through gel-filtration with a Sephadex G25 column equilibratedwith PBS pH 6.5. The conjugate was sterile-filtered using a 0.22 μMMillex-GV filter. The number of drug molecules linked per anti-ErbB2/neu antibody molecule was determined by measuring the absorbance atboth 252 nm and 280 nm of the filtered material. The drug to antibodyratio was found to be about 4.5. The conjugated antibody was furtherbiochemically characterized by size exclusion chromography (SEC) andfound to be over 96% monomer.

Example 12

The stability studies of 3AA-MDC-anti-Erb B/neu antibody were evaluatedin Sprague-Dawley rats. Sprague-Dawley rats were administered 10 mg/kg3AA-MDC-anti-Erb B/neu (based on the antibody component) by tail veininjection. Blood samples were collected from each mouse via thesaphenous vein at 0 h, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h,day 2, day 3, day 4, day 7, day 14, day 21, day 28 after injection.Blood was collected into heparin coated tubes followed by centrifugation(14,000×g, 3 minutes) to isolate plasma. Plasma concentrations of totalAnti-Erb B/neu and antibody-drug conjugates were measured by ELISA.Total antibody concentration in the serum samples was measured asfollows: 96-well ELISA plates were coated with HER2 ECD in 2 μg/mLcarbonate/bicarbonate buffer (pH 9.6) at 4° C. overnight. After removalof the coat solution, nonspecific binding sites were blocked byincubating with blocking solution (PBS, 1% BSA, 0.05% Tween 20) at roomtemperature for 1 hour. The plates were then washed with wash buffer(0.05% Tween in PBS), and standards or samples diluted in PBS wereadded. After a 2 h incubation, plates were washed and mouse anti-humanIgG-horseradish peroxidase conjugate (Sigma, St. Louis, Mo.) was addedfor an additional 2 h. Plates were then washed again. Subsequently, 100μL of 3,3,5,5-tetramethylbenzidine (Sigma, St. Louis, Mo.) were added toeach well, and upon color development, the reaction was stopped with 100μL of 1 N sulfuric acid. Absorbance was measured using a VMax KineticMicroplate reader (Molecular Devices, Sunnyvale, Calif.) at 490 nm. Formeasurement of antibody-drug conjugates concentration, wells were coatedwith HER2 ECD and serum samples added as above. After the 2-h sampleincubation, the plates were washed, rabbit anti-maytansine antibody wasadded to each well, and the plates were incubated for 1 h. Plates werethen washed, and HRP-conjugated goat anti-rabbit IgG (Sigma) was addedfor an additional 1 h incubation. Color detection and measurement wereperformed as described above. Noncompartmental pharmacokineticparameters were calculated with WinNonlin (Pharsight, Mountain View,Calif.). The time-concentration curves of antibody component of3AA-MDC-antibody and the drug component of 3AA-MDC-antibody appeared tofollow bi-exponential declines. The terminal half-life of antibodycomponent of 3AA-MDC-antibody was 571.07, and the terminal half-life ofdrug component of 3AA-MDC-antibody was 88.36 hours.

Example 13 Characterization of Anti-ErbB2 Antibody Drug Conjugate3AA-MDC-Anti-Erb B2 Antibody

The growth inhibitory characteristics of Anti-Erb B2 antibody and3AA-MDC-anti-Erb B2 antibody were evaluated using the Her2 positivebreast tumor cell line, SK-BR-3 (see Hudziak et al. Molec. Cell. Biol.9(3):1165 1172 (1989) and Her2 negative cell line A549 [Shanghai CellCollections, Itd., Co., Shanghai, China]. Briefly, cells were detachedby using 0.25% (vol/vol) trypsin and suspended in complete medium.Aliquots of 100 □L containing 10,000 cells for SK-BR-3 cell line and8,000 cells for A549 cell line were plated into 96-well microdilutionplates. The cells were allowed to adhere overnight at 37° C., and 100 □Lof media containing various concentrations of anti-Erb B2 antibody and3AA-MDC-anti-Erb B2 antibody was then added. After 72 hours, plates werewashed twice with PBS (pH 7.5), and analyzed for relative cellproliferation with CCK-8 reagent (Cell Counting kit-8, Dojindo, Japan).Drug conjugate 3AA-MDC-anti-Erb B2 antibody more significantly inhibitedthe Her2 positive cell proliferation than naked anti-Erb B2 antibody(FIG. 2). Neither naked antibody anti-Erb B2 antibody nor drug conjugate3AA-MDC-anti-Erb B2 antibody inhibited the growth of Her2 negative cellline A549 (FIG. 3).

Example 14 Characterization of Anti-ErbB2 Antibody Drug ConjugateD-Lmcc-Anti-Erb B2 Antibody

The growth inhibitory characteristics of D-Lmcc-anti-Erb B2 antibodywere evaluated using the Her2 positive breast tumor cell line, SK-BR-3(see Hudziak et al. Molec. Cell. Biol. 9(3):1165 1172 (1989) and Her2negative cell line A549. Briefly, cells were detached by using 0.25%(vol/vol) trypsin and suspended in complete medium. Aliquots of 100 □Lcontaining 10,000 cells for SK-BR-3 cell line and 8,000 cells for A549cell line were plated into 96-well microdilution plates. The cells wereallowed to adhere overnight at 37° C., and 100 □L of media containingvarious concentrations of anti-Erb B2 antibody and D-Lmcc-anti-Erb B2antibody was then added. After 72 hours, plates were washed twice withPBS (pH 7.5), and analyzed for relative cell proliferation with CCK-8reagent. Drug conjugate D-Lmcc-anti-Erb B2 antibody strongly inhibitedthe Her2 positive cell proliferation (FIG. 4). Neither naked antibodyanti-Erb B2 antibody nor drug conjugate D-Lmcc-anti-Erb B2 antibodyinhibited the growth of her2 negative cell line A549 (FIG. 5).

Example 15 3AA-MDC-Anti-Erb B2 Antibody Eradicates Human BT474 TumorXenografts

In Vivo Tumor Studies: The effects of 3AA-MDC-anti-Erb B2 antibody onthe growth of established tumors were examined on human BT474 tumorxenografts. Human BT474 cells (ATCC, HTB-20) were cultured in DMEMmedium supplemented with 10% fetal bovine serum, 2 mM glutamine andantibiotics. Female BALB/c nude mice, 8-9 weeks old, were injectedsubcutaneously with 1×10⁷ tumor cells in the dorsal area in a volume of100 μL. When the tumor xenografts reaches a size of 150-200 mm³(calculated as 0.5×(length×width²), animals were then treated withanti-Erb B2 antibody (5 or 15 mg/kg), 3AA-MDC-anti-Erb B2 antibody (5 or15 mg/kg), or control antibody (Rituximab, 15 mg/kg). Animals were dosedevery 3 weeks for a total of 3 doses i.v. in a volume of 100 μL. Eachgroup consisted of 10 mice. Tumor size was determined at 3 daysintervals. 49 days after tumor cell inoculation, animals were euthanizedand tumors were removed and weighed. As shown in FIG. 6, Rapid tumorshrinkage was seen by 3AA-MDC-anti-Erb B2 antibody (15 mg/kg) from day7. From day 10 onwards 3AA-MDC-anti-Erb B2 antibody (15 mg/kg) treatedtumors had shrunken to non-palpable. Compared to the unconjugatedAnti-Erb B2 antibody, 3AA-MDC-anti-Erb B2 antibody more significantlyinhibited the tumor growth as assessed by tumor weight measurements 49days after drug treatment.

Example 16 3AA-MDC-Anti-Erb B2 Antibody Eradicates Human NCI-N87 TumorXenografts

In Vivo Tumor Studies: The effects of 3AA-MDC-anti-Erb B2 antibody onthe growth of established tumors were examined on human NCI-N87 tumorxenografts. Human NCI-N87 cells (ATCC, CRL-5822) were cultured in DMEMmedium supplemented with 10% fetal bovine serum, 2 mM glutamine andantibiotics. Female BALB/c nude mice, 4-8 weeks old, were injectedsubcutaneously with 5×10⁶ tumor cells in the dorsal area in a volume of100 μL. When the tumor xenografts reaches a size of 100-200 mm³(calculated as 0.5×(length×width²)), animals were then treated withAnti-Erb B2 antibody (5 or 15 mg/kg, i.v), 3AA-MDC-anti-Erb B2 antibody(5 or 15 mg/kg, i.v), or control antibody (Rituximab, 15 mg/kg, i.v).Animals were dosed once per week for a total of 5 doses i.v. in a volumeof 100 μL. Anti-Erb B2 antibody treatment of NCI-N87 xenografts wasdiscontinued on day 15 and switched to 3AA-MDC-anti-Erb B2 antibody (5or 15 mg/kg, i.v.) from the day 21 onwards. Each group consisted of 10mice. Tumor size was determined at 3 days intervals. 42 days after tumorcell inoculation, animals were euthanized and tumors were removed andweighed. As shown in FIG. 7, rapid tumor shrinkage was seen by3AA-MDC-anti-Erb B2 antibody (5 or 15 mg/kg) from day 11. From day 32onwards 3AA-MDC-anti-Erb B2 antibody (5 or 15 mg/kg) treated tumors hadshrunken to non-palpable. Compared to the unconjugated anti-Erb B2antibody, at 5 or 15 mg/kg dose tested, 3AA-MDC-anti-Erb B2 antibodymore significantly inhibited the tumor growth as assessed by tumorweight measurements 42 days after drug treatment.

Example 17 Cellular Metabolites of 3AAMDC-Antibody and D-Lmcc-Anti-ErbB2 Antibody

Cellular Metabolites of 3AAMDC-antibody and D-Lmcc-anti-Erb B2 antibodywere assayed as described in Erickson, et al. Cancer Res 66:4426-4433(2006). Briefly, A431 cells (6×10⁶) suspended in 3 mL culture mediumcontaining 3AAMDC-antibody at a concentration of 10⁻⁷ mol/L ofconjugated antibody were incubated at 37° C. for 3 to 30 hours. Thecells and the medium were then separated by centrifugation (2,000×g, 5minutes). The supernatant (3 mL) was chilled on ice, mixed with 4 mLice-cold acetone, and kept at −80° C. for at least 1 hour or untilfurther processing. Precipitated protein was removed by centrifugationat 2,500×g and the supernatants were acidified with 5% acetic acid andevaporated to dryness. The samples were dissolved in 0.12 mL of 20%aqueous CH3CN containing 0.025% trifluoroacetic acid (TFA), aliquots of0.1 mL were submitted to LC-MS (FIG. 8, 9, 10).

1. A compound of Formula Ia:

or a salt thereof, wherein X is hydrogen or halo; Y is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and—C(═O)R⁵; R¹ is selected from the group consisting of hydrogen, —OH,—OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl, —CH₂OH,or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl; R⁶ isC₁-C₆ alkyl, phenyl or benzyl; R⁷ is hydrogen, C₁-C₆ alkyl or an aminoacid side chain; R⁸ is hydrogen or C₁₋₆ alkyl; n is 0, 1, 2, 3, 4, 5, 6,7 or 8; p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; and Anti-HER2is an anti-ERB B2/NEU (HER2) antibody.
 2. The compound of claim 1, whichis a compound of Formula Ib

or a pharmaceutically acceptable salt or solvate thereof.
 3. Thecompound of claim 1, which is a compound of Formula Ic

or a pharmaceutically acceptable salt or solvate thereof, whereinAnti-HER2 is an anti-ERB B2/NEU (HER2) antibody.
 4. The compound ofclaim 3, wherein the compound connected to anti-ERB B2/NEU (HER2)antibody is N2′-deacetyl-N2′-(6-maleimido-1-oxo-hexyl)maytansine). 5.The compound of claim 1, wherein the anti-Erb B2/neu antibody is anantibody comprising trastuzumab or pertuzumab or an equivalent thereof.6. A pharmaceutical composition comprising a compound of claim
 1. 7. Amethod of treating a proliferative, inflammatory or immunologic diseaseor condition in a patient in need thereof comprising administering aneffective amount of a compound of claim
 1. 8. A method of treating aproliferative, inflammatory or immunologic disease or conditioncharacterized by Erb B2/neu positive cells in a patient in need thereofcomprising administering an effective amount of a compound of FormulaIVa:

or a salt thereof, wherein the compound of Formula IVa is generated as aresult of a metabolic chemical reaction following administration of acompound of claim 1, or a pharmaceutically acceptable salt thereof, tothe patient, wherein AA is an amino acid or thiolated amino acid,
 9. Amethod of treating a proliferative, inflammatory or immunologic diseaseor condition characterized by Erb B2/neu positive cells in a patient inneed thereof comprising administering an effective amount of a compoundof Formula IVb:

or a salt thereof, wherein the compound of Formula IVb is generated as aresult of a metabolic chemical reaction following administration of acompound of claim 2, or a pharmaceutically acceptable salt thereof, tothe patient, wherein AA is an amino acid or thiolated amino acid.
 10. Amethod of treating a proliferative, inflammatory or immunologic diseaseor condition characterized by Erb B2/neu positive cells in a patient inneed thereof comprising administering an effective amount of a compoundof Formula IVc:

or a salt thereof, wherein the compound of Formula IVc is generated as aresult of a metabolic chemical reaction following administration of acompound of claim 3, or a pharmaceutically acceptable salt thereof, tothe patient, wherein AA is an amino acid or thiolated amino acid. 11.The method of claim 8, wherein AA is

wherein

represents point of connection to the rest of the molecule.
 12. Thecompound of claim 1, wherein: X is halo; Y is C₁-C₆ alkyl; R¹ ishydrogen; R² is C₁-C₆ alkyl; R³ is methyl; R⁴ is —OH; R⁷ is C₁-C₆ alkyl;and R⁸ is C₁₋₆ alkyl.
 13. The compound of claim 1, wherein: X is Cl; Yis C₁-C₆ alkyl; R¹ is hydrogen; R² is C₁-C₆ alkyl; R³ is methyl; R⁴ is—OH; R⁷ is C₁-C₆ alkyl; and R⁸ is C₁₋₆ alkyl.
 14. A pharmaceuticalcomposition comprising a compound of claim
 13. 15. A method of treatinga proliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of claim
 13. 16. The pharmaceutical composition of claim 6,wherein the pharmaceutical composition is suitable for injection orinfusion.
 17. The pharmaceutical composition of claim 14, wherein thepharmaceutical composition is suitable for injection or infusion.